The calcium hypothesis of Alzheimer's disease (AD) posits that dysregulation of calcium homeostasis is the point of convergence of the many factors risk factors and molecular mechanisms that lead to development of AD and its associated neurodegeneration. A corollary of the calcium hypothesis of AD is that restoration of calcium homeostasis will ameliorate AD pathophysiology and reverse neuronal dysfunction. Surprisingly, despite being nearly 3 decades old, this hypothesis has not been definitively tested in vivo. We propose to directly test the calcium hypothesis by normalizing resting free intracellular calcium levels through exogenous expression of the simple calcium buffer proteins calbindin-D9k and parvalbumin-? in the brains of 5XFAD mice, a model of amyloid pathology in AD. In Aim 1, we will determine if reduced resting free calcium cures the dystrophic axons and neurites that develop around plaques and likely contribute to neuronal dysfunction and increased amyloid generation In Aim 2, we will determine if increasing calcium buffering in the brains of amyloid plaque-containing 5XFAD mice can restore electrophysiological and cognitive impairments. Two adeno-associated viruses will be co-injected into the ventricles of postnatal P0 mouse pups, resulting in viral transduction throughout the brain and lifelong transgene expression. One virus expresses the calcium sensor GCaMP6f from the calmodulin kinase II (CaMKII) promoter, which drives expression in the excitatory neurons of the forebrain. The second virus co-expresses mCherry and a calcium buffer protein (either calbindin-D9k or parvalbumin-?) under the control of a tetracycline-off (TetO) promoter. In mice that express the tetracycline transactivator protein (tTA) under the control of a CaMKII promoter, the calcium buffer protein and mCherry will be expressed in the same population of cells as the calcium sensor GCaMP6f in the absence of the tetracycline analog doxycycline (dox). The pregnant mothers and offspring will be fed dox-containing chow until 6 months of age while the plaques develop. Mice will then be implanted with a cranial window to allow repeated in vivo multiphoton imaging of calcium levels, as measured by GCaMP6f fluorescence, in the dystrophic neurites around plaques. After baseline calcium levels have been measured, we will discontinue the dox diet to induce calcium buffer protein expression and monitor calcium levels and dystrophies by multiphoton microscopy for several weeks. Four weeks after induction, memory performance will be assessed in the Morris water maze, fear conditioning and the Y-maze, hippocampal memory tasks in which the 5XFAD mouse is impaired. At the end of behavioral testing, electrophysiology will be performed to measure the afterhyperpolarization (AHP), which is increased in aged 5XFAD mice and correlates with impaired memory. We predict that increased calcium buffering will ?heal? the dystrophic neurites and improve electrophysiological and cognitive functions, providing strong support for the calcium hypothesis, and validate the use of calcium- reducing therapeutics in AD.